Tissue homeostasis is highly dependent on cell spatial organization and mechanical balance. Cells attach on their microenvironment and exert traction forces via the myosin dependent contraction of their actin cytoskeleton. The level of cell contraction has recently been shown to have dramatic impact on cell physiology. It directs stem cells differentiation (Engler et al., 2006, Cell 126, 677-689). It also promotes cell growth and has been shown to be responsible for tumoral transformation (Paszek et al., 2005, Cancer Cell 8, 241-254). It is thus necessary to develop reliable and easy to employ methods to measure cell contraction level.
The two main methods to measure cell tractions forces are based on cell culture substrate deformation. They both have limitations in substrate fabrication and force analysis.
Among different methods being developed for force measurement, Traction Force Microscopy (TFM) is one of the most used. However, due to its rather complicate data processing step, this technique still remains exclusive to some specialized groups. Albeit all the materials needed to perform TFM are nearly routine equipments and reagents in ordinary biological laboratory.
The classical TFM proposed by Dembo and Wang (1999, Biophys J 76, 2307-2316) was done on poly-acrylamide (PAA) gel. Basically, PAA gel was prepared with fluorescent micro-beads incorporated inside. Then, the gel was activated by chemical crosslinkers (e.g. Sulfo-SANPAH) and coated homogeneously with extra-cellular matrix (ECM) protein to make the gel available for cell adhesion. When cells attached to the gel, due to the traction force exerted by the cell, the soft substrate deformed and thus the beads displaced. By comparing the image of the displaced beads and another image of the original beads position taken after detaching the cell (e.g., by trypsin treatment), one can obtain the displacement field. The traction force could therefore be obtained by solving a displacement-force inverse problem.
Due to the random positioning of the fiducial marker beads, an image of the relaxed beads position, which can only be obtained after detaching the cell, is always required to obtain the displacement field. This prohibits immediate visualization of cell traction. In addition, tracking of randomly positioned beads between stressed and relaxed images inevitably required manually intervention to correct false bead detection and linking which is rather time consuming. This method requires long numerical calculations and case specific regularizations to deduce the traction force field from the gel deformation field. In addition, PAA gel activation with sulfo-SANPAH is a quite variable step resulting in non homogeneous and non reproducible activation of the substrate. The experimental measure of fluorescent beads displacement is highly sensitive to focus drift (Marganski et al., 2003, Methods Enzymol 361, 197-211). Subsequent defects in automated bead tracking lead to large errors in force measurement (Sabass et al., 2008, Biophys J 94, 207-220).
Errors associated to bead detection could be overcome by using micropatterned dots array on the gel surface (Balaban, 2001, Nat Cell Biol 3, 466-472).
A second method, cell culture on micro-fabricated pillars, allows a much simpler and thus faster force calculation (du Roure et al., 2005, Proc Natl Acad Sci USA 102, 2390-2395; Tan et al., 2003, Proc Natl Acad Sci USA 100, 1484-1489). However the substrate requires several non-trivial microfabrication steps. In addition, micropillars do not support solvent dewetting, and substrate topography can affect cell behavior.
With both techniques, cells can move freely on the substrate. Therefore, they adopt every kind of shapes. This absence of geometrical constraints prevents any automated process for cell force measurement. They are therefore not appropriated for large-scale experiments.
Cell shape control using adhesive micropattern is an efficient method to overcome the above-mentioned limitations. Indeed, adhesive micropatterns coated with ECM allow the normalization of individual cell shape and an accurate control of the spatial distribution of focal adhesion and actin cables (Parker et al., 2002, FASEB J 16, 1195-1204; Thery et al., 2006, Cell Motil Cytoskeleton 63, 341-355; Thery et al., 2006, Proc Natl Acad Sci USA 103, 19771-19776). Appropriate geometries can impose stringent orientation constraints to actin assembly, reduce cell-cell variability and simplify the force calculation method by controlling the location of force application.
Micropatterning on PAA gel has been realized with stencils (Parker et al., 2002, supra; Wang et al., 2002, Cell Motil Cytoskeleton 52, 91-106), or microstructured stamps (Engler et al., 2004, Cell 126, 677-689; Tan et al., 2003, supra) but micropattern resolution is relatively low.
In both cases, micropatterning requires several microfabrication steps, making the whole process long and difficult to realize. In addition, pattern geometries that have been tried so far did not provide accurate control of cell force field.
Extraction of force from displacement data still requires non-trivial calculation. In addition to the non-trivial numerical calculation, cell shape and force distribution were highly variable. This makes large scale quantitative analysis impossible. In particular, the force measurement was still made from displacement of beads, thus suffering from the drawbacks mentioned above. Although the cell shape was controlled, the geometries chosen in their work couldn't regularize traction force distribution. Thus, forces were still randomly distributed, and substrate deformation was complex and differed from one cell to another.